Antioxidant and fermentation properties of aqueous solutions of dried algal products from the Boso Peninsula, Japan

Food Bioscience 19 (2017) 85–91

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Antioxidant and fermentation properties of aqueous solutions of dried algal products from the Boso Peninsula, Japan

MARK

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Moemi Takei, Takashi Kuda , Mika Eda, Ayane Shikano, Hajime Takahashi, Bon Kimura Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan

A R T I C L E I N F O

A B S T R A C T

Keywords: Edible algae Antioxidant Lactobacillus plantarum Superoxide anion radical scavenging

The mineral and saccharide contents, and antioxidant properties in aqueous extract solutions of eleven dried algal products obtained from the Boso Peninsula, Japan, were investigated. Potassium content was high in the brown alga Sargassum fusiforme. Polysaccharides content and viscosity were high in the red algae Gloiopeltis furcata, Chondrus ocellotus and C. elatus. Total phenolic compound content, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity, and Fe-reducing power were high in brown algae Eisenia bicyclis, S. fusiforme and the red alga Pyropia sp. Superoxide anion (O2−) radical-scavenging capacity was high in G. furcata, C. ocellotus, C. elatus and the green alga Monostroma nitidum. Lactobacillus plantarum strains isolated from the coast could ferment G. furcata, C. elatus and M. nitidum. The O2− radical-scavenging capacities of the red algae were increased by fermentation. These results suggest that some macroalgal beach-cast brown algae without fermentation and red algae with fermentation can be utilized as natural resources for functional foods.

1. Introduction As of the last decade of the 20th century, there were more than 200 different kinds of marine algae used world-wide; the amount of marine algae collected and cultured world-wide in one year has been reported to be more than 2 million tons dry weight (Zemle-White & Ohno, 1999). Far East Asian countries including China, Japan, Korea, and the Philippines, produce a high quantity of marine algal products (Lüning & Pang, 2003). Among them, brown algae production was the highest in China (about 75% of the world-wide total) and red algae production was the highest in the Philippines (about 31% of the worldwide total). According to the Food and Agriculture Organization of the United Nations (FAO, 2014), about 99% of the farmed marine algae are obtained from just seven Asian countries: China (54%), Indonesia (27%), the Philippines (7%), South Korea (4%), North Korea (2%), Japan (2%), and Malaysia (1%). In 2012, the total production value of the marine algae in aquaculture was about US $6.4 billion (FAO, 2014). In the case of European countries, captured production is high in Norway, France, Ireland, and Iceland, whereas aquaculture production is high in Denmark. From ancient times, various algae have been used as food sources in Japan (Murara & Nakazoe, 2001). For example, the oldest literature relating to the use of edible red alga Pyropia sp (nori) as a tribute was described in the Taiho Ritsuryo Code promulgated in AD 701 (Ichi, 2015). Although nori, Saccharina japonica (Makombu), and Undaria

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Corresponding author. E-mail address: [email protected] (T. Kuda).

http://dx.doi.org/10.1016/j.fbio.2017.06.006 Received 17 April 2017; Received in revised form 7 June 2017; Accepted 14 June 2017 Available online 19 June 2017 2212-4292/ © 2017 Elsevier Ltd. All rights reserved.

pinnatifida (Wakame) are very popular edible algae all over the world, at least fifty more algal species are still used as human food in local, mainly coastal, areas in Japan (Ohno, 2004). Examples of these locales include the Noto Peninsula and the Boso Peninsula in Ishikawa and Chiba Prefectures, which are on the Sea of Japan and the Pacific Ocean, respectively. People in these satoumi areas have a culture of eating various marine algae as food, and have traditionally performed nori farming. However, depending on the season, a large amount of algae, including edible and non-edible, can also be deposited on beaches following storms. In this case, a large amount of unutilized algal beach casts are incinerated and/or transported to a landfill at cost. Not only in East Asian countries, but also in Europe, the US, and other countries, the consumption of several types of marine algae is associated with health benefits (Brown et al., 2014; Fleurence & Levine, 2016). Algae are known to be good sources of minerals, including potassium and magnesium, and several water-soluble dietary fibers, such as alginate, laminaran, and fucoidan (Kuda & Ikemori, 2009; Nakata, Kyoui, Takahashi, Kimura, & Kuda, 2016); they also contain other chemical compounds, including phenolic compounds. Reports of antioxidant activities in edible algae have increased recently (Balboa, Conde, Moure, Falqué, & Domínguez, 2013). It has been well reported that reactive oxygen species correlate with inflammation (Mittal, Siddiqui, Tran, Reddy, & Malik, 2014), cancer (Waris & Ahsan, 2006), and ageing (Ludovico & Burhans, 2013). For this reason, there have been many reports about the presence of antioxidants in foods

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M. Takei et al.

washed ashore on Boso Peninsula beaches. These algae were therefore collected from beach casts. M. nitidum (Hitoegusa) and Ulva sp. (Aosa) were collected from the intertidal zone. Pyropia sp. (Amanori) was obtained from an aquafarm. These fresh algae were all dried under the sun at the coast. The dried samples were milled using a blender (Oster 16 Speed Blender; Osaka Chemical Co., Japan) and sieved through a 1-mm2 mesh. The algae powder (5 g) was added to 200 mL of distilled water and heated at 105 °C for 15 min using an autoclave. After cooling with tap water, the algae suspension was centrifuged at 3000 × g for 10 min at 4 °C. The collected supernatant was used as the algal aqueous extract solutions (AES) and stored at − 30 °C until used for analysis.

(Lobo, Patil, Phatak, & Chandra, 2010). In our previous studies, the antioxidant capacities of several traditional algal products obtained from the Noto Peninsula coasts were reported (Eda, Kuda, Kataoka, Takahashi, & Kimura, 2016; Kuda et al., 2016). Furthermore, fermentation with lactic acid bacteria (LAB) has been reported to increase superoxide anion (O2−) radical-scavenging capacity in several food materials, including algae (Nemoto et al., 2017). In some LAB species, compared with the strains isolated from dairy products, several isolates obtained from algal beach casts or fish intestines showed a higher resistance against salt, bile, and acid (Kawahara et al., 2015). In the present study, we sought to examine the beneficial properties of hot aqueous extract solution (AES) of edible algae obtained from the Boso Peninsula, which has species of algae that are different from those of the Noto Peninsula. In particular the mineral, polysaccharide, and antioxidant properties of the AES were measured. Furthermore, the fermentation ability of LABs isolated from this coastal area and the antioxidant properties of the algal extracts after fermentation were determined.

2.3. Determination of minerals in the sample solutions Among the major five cations (Na+, K+, NH4+, Ca2+, and Mg2+), and three anions (Cl-, PO4,3- and SO42-), K+, NH4+, Ca2+, Cl-, PO43, and SO42- were measured using commercially available kits from the series of Reagent Set for Water Analyzer (LR-K, LR-NH4-A, LR-Ca-B, LR-Cl, LR-PO4 and LR-SO4) respectively (Kyoritsu Chemical-Check Lab., Corp., Tokyo, Japan). Na+ was measured using a Na+-ion meter (LAQUA Twin B-721, Horiba, Kyoto, Tokyo). Mg2+ was determined using a diagnosis commercial kit (Magnesium B-Test Wako, Wako Pure Chemical).

2. Materials and methods 2.1. Chemicals Folin-Ciocalteu's phenol reagent, the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical phenazine methosulfate (PMS), 3-(2-pyridyl)-5,6-di(p-sulfophenyl)1,2,4-triazine disodium salt (ferrozine), βnicotinamide adenine dinucleotide (NADH), and nitroblue tetrazolium salt (NBT) were from Sigma-Aldrich (St. Louis, MO, USA). Phloroglucinol dehydrate (PG), potassium ferricyanide, trichloroacetic acid (TCA), FeCl3 and bile (Oxgall) were from Wako Chemicals (Osaka, Japan), while 1,10-phenanthroline was from Nacalai Tesque (Kyoto, Japan). The other reagents were of analytical grade.

2.4. Saccharide contents and viscosity of the AES The total water-soluble saccharide and polysaccharide contents were determined by the phenol–sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) and the alcohol precipitation method (Kuda, Goto, Yokoyama, & Fujii, 1998). The relative viscosity of the sample solutions was directly determined using an oscillation viscometer (Viscomate VM-1G; Yamaichi Electronics; Osaka, Japan) under ice-cold conditions (Kuda & Ikemori, 2009). The relative viscosity was calculated as the quotient of the AES viscosity divided by the distilled water viscosity.

2.2. Preparation of aqueous extract solutions from dried algal solutions A total of eleven dried products (Table 1) from two species of brown algae Phaephyta, Eisenia bicyclis (Arame: E1 and E2) and Sargassum fusiforme (Hijiki: S), five species of red algae Rhodphyta Pyropia sp. (Amanori: P), Gloiopeltis furcata, (Funori: Gf1 and Gf2), Chondrus ocellatus (Tsunomata: Co), C. elatus (Kotoji-tusunomata: Ce), Gelidiaser sp. (Tengusa: Ge), as well as two species of green algae Chlorphyta Monostroma nitidum (Hitoegusa: M) and Ulva sp.(Aosa: U) were obtained from Suzuki Nori Co., Choshi, Chiba, Japan. Among these algal samples, E. bicyclis (Arame), G. furcata (Funori), C. ocellatus (Tsunomata), and C. etatus (Kotoji-tsunomata) are predominantly seaweeds

2.5. Phenolic content and antioxidant properties Total phenolic content, as the polyphenol content in the AES, was determined by the Folin-Ciocalteu method, as described previously (Eda et al., 2016). DPPH radical-scavenging capacity was determined as described previously (Kuda & Ikemori, 2009). Briefly, the diluted sample (0.1 mL) and ethanol (0.1 mL) were put into a 96-well microplate, and

Table 1 Cations and anions in aqueous extract solutions (AES) of dried algal products (mmol/L). Name of algae

Cations

Anions

Scientific

Japanese

Abbreviation

Na

K

NH4

Ca

Mg

K/Na

Cl

PO4

SO4

Phaeophyta Eisenia bicyclis

Arame

E1 E2 S

17.1 39.4 25.7

26.6 26.5 110.8

1.98 1.53 0.28

14.7 14 20

26.2 24.5 23.7

1.56 0.67 4.31

0.52 1.62 4.06

0.08 0.12 0.06

11.9 – 0.8

Tsunomata Kotoji-tsunomata Tengusa

P Gf1 Gf2 Co Ce Ge

8.6 18.8 15.4 46.2 32.5 6.8

2.3 – 1.6 2 11.5 –

2.39 0.25 0.02 1.69 1.71 0.46

7.7 10.9 19.1 13.2 10.8 7.8

20.5 22.0 24.9 6.54 5.21 26.4

0.27 – 0.10 0.04 0.35 –

– 0.84 0.18 4.24 2.97 0.01

0.36 0.07 0.04 0.05 0.06 0.05

– – 54.0 1.7 – 1.3

Hitoegusa Aosa

M U

30.8 27.4

0.1 0.8

0.08 1.51

19.4 42.2

26.5 29.8

0.00 0.03

1.35 0.45

0.04 0.08

1.0 63.8

Sargassum fusiforme Rhodphyta Pyropia sp. Gloiopeltis furcata Chondrus ocellatus Chondrus elatus Gelidiaceae Chlorophyceae Monostroma nitidum Ulvaceae

Hijiki Amanori Funori

Values are mean of triplicate measurement.

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measured as described above.

absorbance at 517 nm (Abs1) was measured using a microplate reader (SH-1000 Lab). Next, 1 mmol/L DPPH radical (0.025 mL) was added and incubated at 37 °C for 30 min and the absorbance (Abs2) was measured again. The DPPH radical-scavenging capacity was calculated using the following formula:

2.8. Statistical analysis The antioxidant activities and phenolic content of the algal AES data are presented as means and standard errors (n = 3). Data pertaining to the antioxidant capacities before, versus after, fermentation were subjected to Dunnett's post hoc tests, using statistical software (Excel Statistic Ver. 6, Esumi, Tokyo, Japan). Significant differences were accepted at p < 0.05.

Abs2 : sample − Abs1 : sample ⎞ × 100 Radical scavenging capacity(%) = ⎛1 − Abs2: control − Abs1 : control ⎠ ⎝

O2− radical-scavenging activity was measured by a non-enzymatic method (Kuda & Ikemori, 2009). The sample solution (0.01 mL) was treated with 0.05 mL of 250 mmol/L phosphate buffer (pH 7.2), 2 mmol/L NADH (0.025 mL), and 0.5 mmol/L NBT (0.025 mL), while absorbance at 560 nm (Abs1) was measured as a blank value. After five min incubation at ambient temperature with 0.025 mL of 0.03 mmol/L PMS, the absorbance (Abs2) was measured again. The radical-scavenging capacity was calculated using the above formula. Ferrous-reducing power was determined as described in our previous report (Eda et al., 2016). For each 0.05 mL of the sample solution, 0.025 mL of 0.1 mol/L phosphate buffer (pH 7.2), and 0.025 mL of 10 g/L potassium ferricyanide were placed in a 96-well microplate. After incubation at 37 °C for 60 min, 0.025 mL of 10% TCA and 0.1 mL of distilled water were added and the absorbance was measured at 700 nm (Abs1). Next, 0.025 mL of 0.1% FeCl3 was added to the mixture and the absorbance was measured again (Abs2). Ferrous reducing power was calculated using the following formula:

3. Results and discussion 3.1. Mineral composition The ratio of K to Na (K/Na) is regarded to be important for people who take diuretics to control hypertension and who suffer from excessive excretion of potassium (Adrogu & Madias, 2007; Perez & Chang, 2014). Minerals, particularly Ca and Mg are also important as constituents of bones, teeth, soft tissues, hemoglobin, muscle, blood, and nerve cells, and are vital for overall mental and physical wellbeing (Orchard et al., 2014; Tai, Leung, Grey, Reid, & Bolland, 2015). The mineral composition of the sample solutions is shown in Table 1. The solution of S. fusiforme (Hijiki: S), a brown alga, showed the highest K ion content (111 mmol/L) as well as the highest K/Na (4.3). Solutions of the other brown algae, E. bicyclis (Arame: E1 and E2) also contained a high K ion content, being about 27 mmol/L each. Mg and Ca ions were also high in the brown algae, being about 24–26 mmol/L and 14–20 mmol/L, respectively. In the case of red algae, although the mineral composition varied, the K ion content was also high, being about 12 mmol/L in C. elatus (Kotoji-tsunomata: Ce) solution. Two green algae: M. nitidum (Hitoegusa: M) and Ulva sp. (Aosa: U) had both high Mg and Ca ion contents, being about 27–30 mmol/L and 19–42 mmol/L, respectively.

Reducing power (OD 700 nm) = (Abs2 of sample − Abs1 of sample) −(Abs2 of control − Abs1 of control)

2.6. Isolation of Boso satoumi LAB strains Eight fresh algal beach casts, eight samples of beach sands or soils, and eight fish intestinal contents were collected from the Boso Peninsula between August 2015 and July 2016. Approximately 3 g of each sample was inoculated into 25 mL of de Man, Rogosa, and Sharpe (MRS) broth (Oxoid, Basingstoke, UK) and incubated at 30 °C for 3 days. After incubation, a loop-full of the turbid culture was streaked onto an MRS agar plate (Oxoid) and incubated anaerobically at 30 °C under an AnaeroPack system (Mitsubishi Gas Chemical, Tokyo, Japan) for three days. A typical colony from each MRS plate was isolated. After selection for being catalase negative, Gram staining and cell morphology were evaluated. Gram-positive and catalase-negative rods and cocci were identified as LAB rods and LAB cocci. From these LAB isolates, three LAB strains were selected based on their tolerance to 1% bile and acid (pH 5), and lactose utilization (Kawahara et al., 2015). The selected isolates were identified based on 16S rRNA gene sequencing. Following amplification of the 16S rRNA gene, using the PCR primers 27F and 1492R, the PCR product was sequenced by Macrogen Japan Corp. (Kyoto, Japan). The homology search was performed using blastn of the DNA Data Bank of Japan (http://ddbj.nig.ac.jp/blast/blastn). To determine the bacterial carbohydrate utilizing properties, the fermentation pattern of the three isolates was examined using the API 50 CHL system (BioMereux, MarcyI′Etoile, France).

3.2. Relative viscosity and water-soluble polysaccharides As shown in Fig. 1A, the relative viscosity was low in brown and green algal AES. With the exception of Pyropia sp. (Amanori: P), the

2.7. Fermentation by Boso satoumi LAB isolates The three Boso satoumi strains were stored using ceramic beads (Microbank, Iwaki Co., Ltd., Tokyo, Japan) at − 80 °C. A bead of each strain was inoculated into 5 mL of MRS broth. After 48 h incubation at 30 °C, these cultures were used for algal fermentation. The pre-cultured strains (0.03 mL) were inoculated into 3 mL of the AES and incubated at 30 °C for 5 days. If turbidity could be observed by the naked eye, the pH was determined using a pH meter (LAQUA Twin B-711, Horiba). The phenolic content and antioxidant properties of the fermented AES were

Fig. 1. Relative viscosity (A) and water soluble saccharides (B) in aqueous extract solutions (AES) of dried algae. CM: the AES was gelled and the viscosity could not be measured. (B): Total (closed columns), poly- (semi-closed columns) and low molecularweight- (open columns) carbohydrates were assayed by the phenol-sulfuric acid method. Values are mean and standard error of the mean (n = 3).

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3.4. Antioxidant properties

relative viscosity of red algae AES was very high, being 50 or greater, particularly for C. elatus (Kotoji-tsunomata: Ce), which had a relative viscosity of 340. In the case of Gelidaseae sp. (Tengusa, Ge), the AES was gelled and the viscosity could not be measured. Fig. 1B shows the total, poly-, and low-molecular-weight saccharide content in the AES. Although it is often thought that relative viscosity is highly correlated with polysaccharide content, it is clear that the polysaccharide content in the red algal AES was not correlated with the relative viscosity value. Furthermore, the M. nitidum (Hitoegusa: M) solution had the highest polysaccharide and low molecular-weight polysaccharide contents although its relative viscosity was low. The main viscous polysaccharides in G. furcata (Funori) are the funorans, and in Chondrus sp. (Tsumomata) are the carrageenans (Necas & Bartosikova, 2013; Tuvikene et al., 2015). The biological activity of carrageenans has been well described (Liu, Zhan, Wan, Wang, & Wang, 2016). There are some reports on the properties of funorans, they are also functional-foods having anti-tumor and immunemodifying activities (Ren, Wang, Noda, Amano, & Ogawa, 1995).

3.4.1. DPPH radical-scavenging capacity DPPH has been used extensively as a free radical to evaluate the reduction of substances in various foods, including edible algae, because it is simple and affordable (Cian, Garzón, Ancona, Guerrero, & Drago, 2015). The percentages of DPPH radical-scavenging activity for the different AES at a concentration of 0.5 mL AES/mL are shown in Fig. 2B. Among the eleven algal AES samples, the DPPH radical-scavenging activity was high in the AES from E. bicyclis (Arame: E1 and E2) and Pyropia sp. (Amanori: P). In general, the order of scavenging activity appeared to agree with the phenolic content (Fig. 2A), although the capacity of the red algae G. furcata (Funori: Gf1, Gf2) was lower than expected. 3.4.2. O2− radical-scavenging capacity The O2− radical-scavenging capacity of the AES was confirmed using the non-enzymatic NBT method. The individual capacities at a concentration of 0.015 mL AES/mL are shown in Fig. 2C. AES from red and green algae had a much higher O2− radical-scavenging capacity compared to those from brown algae. That data did not agree with the phenolic compound content and DPPH radical-scavenging capacity data. In fact, it appears to agree better with total saccharide content (Fig. 1B). Among the red and green algae, G. furcata (Funori: Gf1, Gf2), Gelidiaser sp. (Tengusa: Ge), C. elatus (Kotoji-tsunomata: Ce), and M. nitidum (Hitoegusa: M) had the highest O2− radical-scavenging capacities. In most organisms, O2− radicals are converted to hydrogen peroxide by superoxide dismutase (Kawano et al., 2015). In the absence of transition metal ions, hydrogen peroxide is stable. However, hydroxyl radicals can be formed by the reaction of O2− with hydrogen peroxide in the presence of metal ions, usually ferrous or copper (Pisoschi & Pop, 2015). Hydroxyl free radicals are much more reactive and toxic than O2−. The O2− radical-scavenging activity of the AES (shown in Fig. 2C) suggests that the edible algae, even the red and green algae that had low DPPH radical-scavenging capacities, have the benefits of being able to scavenge not only O2−, but also hydrogen peroxide and hydroxyl radicals.

3.3. Total phenolic content The total phenolic contents in the AES prepared from the dried algae products are shown in Fig. 2A. The phenolic concentrations in the AES from E. bicyclis (Arame: E1 and E2), S. fusiforme (Hijiki: S), Pyropia sp. (Amanori: P), and G. furcata (Funori: Gf1, Gf2) were high, ranging from approximately 0.6–0.8 mmol PG equivalent (PGEq)/L. C. elatus (Kotojitsunomata: Ce), Gelidiaser sp. (Tengusa: Ge), and the AES from the two green algae M. nitidum (Hitoegusa: M) and Ulva sp.(Aosa: U) had a phenolic content less than 0.2 mmol PGEq/L. The total phenolic content of Pyropia sp. (Amanori: P) and G. furcata (Funori: Gf1, Gf2) was similar to that of a previous report (Eda et al., 2016; Nemoto et al., 2017). However, the phenolic content of E. bicyclis (Arame: E1 and E2) and S. fusiforme (Hijiki: S) AES was far lower than that reported for the brown algae Ecklonia sp. (Kuda & Ikemori, 2009; Kuda et al., 2016). There are several known bioactive compounds in these brown algae, particularly phlorotannins, that function as strong antioxidants (Boi, Cuong, & Vinh, 2017; Eom, Kim, & Kim, 2012).

A: Total phenolic compounds Scavenging %

mmol PGEq*/L

1.0 0.8 0.6 0.4 0.2

0.0 E1 E2 S

60 40

20

60 40 20 E1 E2 S

B: DPPH radical scavenging

80

80

0

M U

OD at 700nm

Scavenging %

100

P Gf1Gf2 Co Ce Ge

100

C: O2- radical scavenging

1.0

P Gf2 Gf1 Co Ce Ge

M U

D: Fe-reducing power

0.8 0.6 0.4 0.2 0.0

0 E1 E2 S

P Gf1Gf2 Co Ce Ge

Brown algae

Red algae

Fig. 2. Total phenolic compound content (A), DPPH and = 3).

O2-

E1 E2 S

M U

Brown algae

Green algae

P Gf1 Gf2 Co Ce Ge

Red algae

M U

Green algae

radical scavenging capacities (B, C) and Fe-reducing power (D) of the AES. Values are mean and standard error of the mean (n

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Consistent with this, AES from the brown algae obtained from the Noto Peninsula could not be fermented (Kuda et al., 2016).

3.4.3. Ferrous-reducing power Most non-enzymatic antioxidant activities such as the scavenging of free radicals and the inhibition of peroxidation are mediated by redox reactions (Lobo et al., 2010). Compounds with reducing power are electron donors that can reduce the oxidized intermediates of lipid peroxidation processes and thereby act as primary and secondary antioxidants (Anderson, Cameron, Murphy, & Tuttle, 2016). Fig. 2D shows the ferrous-reducing power assessed at a concentration of 0.5 mL AES/mL. The highest ferrous-reducing power was obtained in the AES from E. bicyclis (Arame: E2), followed by those from S. fusiforme (Hijiki: S), E. bicyclis (Arame: E1 and E2), and then Pyropia sp. (Amanori: P), which supported the phenolic content and DPPH radicalscavenging data. The phenolic content and antioxidant capacity data for S. fusiforme (Hijiki: S), Pyropia sp. (Amanori: P), G. furcata (Funori: Gf1, Gf2), and Gelidiaser sp. (Tengusa: Ge) agreed with those from our previous studies (Kuda & Ikemori, 2009). The data for E. bicyclis (Arame), C. ocellatus (Tsunomata), C. elatus (Kotoji-tsunomata), and M. nitidum (Hitoegusa) are presented for the first time in this study.

3.6. Antioxidant properties of the fermented aqueous solutions The phenolic content, DPPH radical-scavenging capacity, and Fereducing power were not changed by LAB fermentation. On the other hand, O2− radical-scavenging capacity of the three red algae AES was clearly increased by LAB fermentation, particularly by L. plantarum Boso-SU6 fermentation (Fig. 4A–C). A previous study has shown that the AES from G. furcata (Funori, obtained from Sanriku in the north east of Japan) could be fermented by L. plantarum (Nemoto et al., 2017). The fermentation increased the O2− radical-scavenging capacity of the AES. In this previous study, the O2− radical-scavenging capacity fractionated along with high molecular-weight compounds (> 300 kDa). This study is the first to report fermentation of C. elatus (Kotoji-tsunomata) and M. nitidum (Hitoegusa). Other than algae, increasing the O2− radical-scavenging capacity of milk, soy milk, and Japanese white radish by fermentation with L. plantarum have been reported (Kuda, Kaneko, Yano, and Mori (2010). It has also been reported that several foods fermented and activated using L. plantarum have anti-inflammatory effects in vitro and in vivo (Kondo et al., 2016; Nemoto et al., 2017). Furthermore, some selected L. plantarum strains have anti-infective and anti-inflammatory activities (Hirano et al., 2017). The results of this study suggest that fermented G. furcata (Funori) and C. elatus (Kotoji-tsunomata) are promising functional food materials, which are capable of protecting against reactive oxygen species. On the other hand, AES from the brown algae E. bicyclis (Arame) and S. fusiforme (Hijiki) could not be fermented, although the AES have a high potassium content and high antioxidant capacities in the DPPH radical and Fe-reduction assays. E. bicyclis (Arame), G. furcata (Funori), C. ocellatus (Tsunomata) and C. elatus (Kotoji-tsunomata) are the principal washed-ashore seaweeds found on Boso Peninsula beaches. Although it is considered that study of the drying and heating methods are needed. It is hoped that these macroalgal beach-casts with, or without, fermentation can be utilized as natural resources for the production of functional foods, cosmetics, and in medical applications, instead of being processed for incineration or delivery to the landfill. Some fermented foods are improved using co-cultures rather than single starters (Kondo et al., 2016). To improve the functional properties of the brown algae, a fermentation study using a co-culture system is now in progress.

3.5. Fermentation properties In this study, all three of the selected LAB strains (Boso-SU4, -SU5 and -SU6 isolated from soil, sands and intestine of thread-sail filefish Stephanolepis cirrhifer, respectively) were identified as Lactobacillus plantarum by 16S rRNA gene sequencing and BLAST analysis. As shown in Table 2, these three isolates, particularly L. plantarum Boso-SU6, could ferment a wide range of different carbohydrates compared to their type strain. All three strains could lower the pH value of G. furcata (Funori: Gf1, Gf2), C. elatus (Kotoji-tsunomata: Ce), and M. nitidum (Hitoegusa: M) AES after 2–5 days of fermentation (Fig. 3A–D). L. plantarum Boso-SU6 showed the strongest activity toward the three red algae AES (Fig. 3A–C). On the other hand, the AES of E. bicyclis (Arame), S. fusiforme (Hijiki), Pyropia sp. (Amanori), C. ocellatus (Tsunomata), and Ulva sp. (Aosa) could not be fermented by these LAB strains. Some brown algae that contain high levels of phenolic compounds, such as Ecklonia kurome, have been reported to have bactericidal effects, particularly on gram-positive bacteria (Eom et al., 2012). Table 2 Carbohydrate utilization by Lactobacillus plantarum strains isolated from the Boso Peninsula coast.

Carbohydrates

Type NBRC101974

L-Arabinose

Boso-SU4 LC144973a

Boso-SU5 LC194506

+

+ + + +

L-Sorbose

L-Rhamnose Methyl-α-Dmannopyranoside N-Acetyl glucosamine Arbutin D-Cellobiose D-Raffinose D-Turranose D-Arabitol Gluconate

Boso-SU6 LC194507

+ +

+

+

+

+ +

+ + +

+ + + +

+ + +

+ +

4. Conclusion In this study, we examined the minerals, water-soluble polysaccharide, and antioxidant properties of hot water extract solutions (AES) from eleven algal dried products obtained from the Boso Peninsula, Japan. Among the AES, the brown algae E. bicyclis and S. fusiforme AES had a high content of potassium. Water-soluble polysaccharide content and viscosity were high in the red algae G. furcata, C. ocellatas, and C. elatus AES. E. bicyclis, S. fusiforme and Pyropia sp. AES had high total phenolic compound contents, strong DPPH radicalscavenging activities and ferrous-reducing powers. On the other hand, the AES of the red algae G. furcata, C. ocellatas, and C. elatus and the green alga M. nitidum had a high O2− radical-scavenging activity. The AES of four algal products could be fermented by the selected L. plantarum strains isolated from the Boso Peninsula. The O2− radicalscavenging activity of the red algae AES was increased by LAB fermentation. Since E. bicyclis, G. furcata, C. ocellatas, and C. elatus are the principal seaweeds washed ashore on Boso Peninsula beaches, these results suggest that these macroalgal beach-casts, with or without fermentation, can be utilized as natural resources for functional foods.

+

All strains fermented D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-sorbitol, Dmannitol,amygdalin, esculin, salycin, D-maltose, D-lactose, D-melibiose, D-sucrose, D-trehalose, D-melezitose, and gentiobiose, None of the strains fermented glycerol, erythritol, D-arabinose, D-xylose, L-xylose, D-adonitol, Methyl-β-D-xylopyranoside, dulcitol, inositol, methyl-α-D-glucopyranside, inulin, starch, glycogen, xylitol, D-lyxsose, D-tagatose, D-fucose, L-fucose, L-arabitol, 2-keto gluconate, and 5-keto gluconate. a Accession numbers.

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B

A 7

G. frucata (Gf1)

7

C G. frucata (Gf2)

7

D C. elatus

6

6

SU5 6

SU5

SU5

SU4

5

5 4

0

1

2

3

4

5

6

5

5

4

4

SU6

SU6

3

3

M. nidum

SU6

4

SU6

7

0

1

2

3

4

5

0

1

2

3

4

5

SU4 0

1

2

3

4

5

Fig. 3. Values of pH in aqueous extract solutions of G. furcata (Funori: A, B), C. elatus (Kotoji-tsunomata: C) and M. nitidum (Hitoegusa: D) fermented by L. plantarum strains. Values are mean and standard error of the mean (n = 3). Fig. 4. O2- radical scavenging activity of G. furcata (Funori: A, B), C. elatus (Kotoji-tsunomata: C) and M. nitidum (Hitoegusa: D) without fermentation (control: open circles) and fermented with L. plantarum Boso-SU4 (closed circles), -SU5 (triangles) and -SU6 (squares). Values are mean and standard error of the mean (n = 3). * Significant difference from control AES (< 0.05).

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